In this study, we report for the first time the presence of an antidiuretic factor in the head of the Colorado potato beetle (Leptinotarsa decemlineata (Say)) which acts directly on Malpighian tubules. Biologically active fractions were isolated from the head and separated using molecular weight filtration and high-performance liquid chromatography (HPLC). The resulting fractions were tested for their antidiuretic activity on single isolated Malpighian tubules. Antidiuretic activity was found in the 25% acetonitrile Sep-Pak fraction and the Cn-2 (3000-10,000 MW) and Cn-3 (<3000 MW) fractions, suggesting that the antidiuretic factor was probably a peptide of 25 to 50 amino acids. The antidiuretic factor was very potent, since after five successive fractionations on two different HPLC columns, a high level of inhibition (63%) of fluid secretion by Malpighian tubules could be observed at low dose (0.14 head-equivalent/microl). The antidiuretic factor isolated from the head of the Colorado potato beetle was not affected by repeated freezing and thawing but was sensitive to heat. The differences observed between the Colorado potato beetle antidiuretic factor and other insect diuretic and antidiuretic factors may indicate the possibility of a novel family of water regulation hormones in insects.